Synthetic biology brings together the disciplines of engineering, biology and bioinformatics. Its focus is to make the engineering of biology easier and more predictable. The basis of synthetic biology is the production of genetic pathways using nucleic acid sequences as “building blocks”. Technological applications of synthetic biology include the production of biofuels, environmentally friendly chemicals, drugs and new materials.
In order to meet the challenge of combinatorial assembly of many genetic pathways in parallel at a reasonable cost, it is necessary to re-think typical approaches for assembly of nucleic acids. For pathways containing a large number of genes and associated regulation and control elements, existing pairwise, hierarchical assembly approaches require a significant number of assembly stages that render the approaches impractical. For instance, a pathway with 30 components (e.g., 10 genes with associated regulation and control elements) would require 5 rounds of hierarchical assembly. In order to make hundreds or thousands of such pathways, the liquid handling alone needed between each round of assembly would render the approach impractical.
The BioBricks™ system (Cambridge, Mass.) allows for the assembly of up to 3 “parts” or nucleic acid sequences (typically genes and associated regulation and control elements) at once by making use of standardised parts and restriction enzymes.
Gibson et al (Nature Methods 6(5): 343-345, 2009) relates to a method for enzymatic assembly of DNA molecules of up to several hundred kilobases. The method is an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase. The method therefore requires a number of different enzymes and requires the DNA molecules that are to be assembled to have a long stretch of overlapping sequence. In practice, this means that the DNA molecules need to be made de novo for each assembly.
Ellis et al (Nature Biotechnology 27(5): 465-471. 2009) relates to diversity-based, model guided construction of gene networks with predicted functions. The approach couples libraries of diversified components (synthesizes with randomized nonessential sequence) with in silico modelling.
There is therefore a need in the art for a method for combinatorial assembly of nucleic acid sequences such as genes and associated regulation and control elements that allows for the fast and reliable construction of large genetic pathways using minimal reagents.